Subarachnoid hemorrhage (SAH) is a type of stroke associated with high morbidity and mortality, and survivors often experience severe sequelae. Various brain injuries that occur within 72 h after SAH are referred to as early brain injuries (EBIs), and these injuries are critical factors that affect the prognosis of patients. SAH results in various intracranial physiological changes, including hypoxia, ion disorder, oxidative stress, inflammation, and electrophysiological disorder. Neuronal injury following different degrees of damage can lead to apoptosis, necrosis, and other cell death processes, ultimately resulting in neurological deficits and even death of individuals (Sehba et al., 2012).

The integrated stress response (ISR) system is an evolutionarily conserved intracellular signaling network that helps maintain cellular proteostasis. Phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) is a central process of ISR. Phosphorylated eIF2α (P-eIF2α) can inhibit protein synthesis and induce the expression of certain stress-related proteins. However, when external stimuli are strong or prolonged, excessive P-eIF2α induces the upregulation of CHOP expression, which acts as a switch for cell apoptosis (Costa-Mattioli and Walter, 2020). CHOP can inhibit the expression of Bcl-2 and induce the translocation of Bax to mitochondria. CHOP also increases ATP consumption and reactive oxygen species production (Song and Kim, 2017; Tsukano et al., 2010; Tuzlak et al., 2016). Previous studies have shown that the eIF2α/CHOP pathway is involved in mediating neuronal apoptosis after SAH (Fan et al., 2017). Furthermore, siRNA-mediated inhibition of CHOP expression reduces neuronal loss and EBI after SAH and improves functional performance in rats (He et al., 2012).

The ubiquitin-proteasome system (UPS) plays an important role in maintaining proteostasis (Meyer-Schwesinger, 2019). The UPS degrades approximately 80% of proteins in cells, including damaged, oxidized, and misfolded/unfolded proteins (Ciechanover, 2005; Yang et al., 2021). Unfolded proteins are specifically recognized by GRP78 in the endoplasmic reticulum membrane. GRP78 signals the presence of unfolded proteins to PERK, which subsequently activates the eIF2α/CHOP apoptosis pathway through phosphorylation (Bertolotti et al., 2000; Carrara et al., 2015; Walter and Ron, 2011). The impairment of proteasome function causes the aggregation and deposition of proteins in the cell. In the central nervous system, the accumulation of abnormal protein polymers has a neurotoxic effect and can lead to neuronal damage and even apoptosis (Sun et al., 2007). The proteasomal degradation of proteins requires tagging with a lysine 48 (K48)-linked ubiquitin chain (Samant et al., 2018). Therefore, ubiquitin-positive intraneuronal inclusions are a pathological feature of many neurodegenerative diseases (Ross and Pickart, 2004). Although proteasomes are involved in the regulation of many biological processes in the nervous system (Lehman, 2009), no relevant studies have been conducted on their role in SAH.

p97/VCP (valosin-containing protein) is an ATPase that belongs to the AAA protein family. p97 is considered an additional component of the UPS (Meyer et al., 2012). In the ubiquitin-dependent protein degradation process, p97 plays a regulatory role in several reaction processes, thus facilitating the smooth progression of the entire degradation process. p97 can help the proteasome recruit ubiquitinated proteins, extend the ubiquitination chain of proteins or reduce the degree of ubiquitination of complex proteins, promote the recognition of ubiquitinated proteins by the proteasome, and help the proteasome develop the spatial structure of ubiquitinated proteins (Beskow et al., 2009; Jentsch and Rumpf, 2007; Rumpf and Jentsch, 2006; Wang et al., 2006). Along with the proteasome, p97 maintains proteostasis in cells and is essential for normal metabolic activities. p97 plays a role in intracellular nuclear membrane reconstruction, membrane fusion, cell cycle regulation, endoplasmic reticulum associated degradation, autophagy, and DNA repair (Hetzer et al., 2001; Kondo et al., 1997; Lu et al., 2022; Partridge et al., 2003; Rabinovich et al., 2002). Previous studies have shown that p97 exerts an important antiapoptotic effect. Transfection of cells for p97 overexpression led to increased cell proliferation. However, the inhibition of p97 expression had the opposite effect, resulting in cell death (Duscharla et al., 2018). The impairment of p97 function can lead to the interruption of protein ubiquitination and degradation, resulting in cell death and the development of various neurodegenerative diseases (Hirabayashi et al., 2001; Watts et al., 2004). In a previous study, researchers performed a conditional knockout of p97 in mice and found that the brain tissue of mice exhibited pathological changes, including neuronal loss and brain atrophy (Wani et al., 2021). This finding indicates that p97 has an important role in neurons.

As mentioned above, p97 shows an antiapoptotic effect and is essential for the proper functioning of the nervous system. It can maintain proteostasis by promoting the function of proteasomal protein degradation. The ISR system is the most important feedback system during the disruption of proteostasis, and the activation of the ISR system is associated with neuronal apoptosis after SAH. Therefore, prior to our experiment, we hypothesized that overexpression of p97 can enhance proteasome function and further inhibit the activation of the eIF2α/CHOP pathway of ISR, thereby promoting the survival of neurons after SAH. In the present study, we investigated the role of p97 in neuronal apoptosis induced after SAH and determined the underlying mechanism.